蛋白质胰蛋白酶水解过程双位点漏切肽段的质谱鉴定

为说明胰蛋白酶漏切位点数目设置对蛋白质搜库结果的影响，采用基质辅助激光解吸电离 串联飞行时间质谱(MALDI-TOF/TOF)高置信度地鉴定了细胞色素C和大豆脱落胁迫成熟蛋白。在细胞色素C和大豆脱落胁迫成熟蛋白的胰酶水解液中分别发现了2个和4个双胰酶漏切位点肽段，确认了文献中报道的当赖氨酸和赖氨酸相邻或赖氨酸、精氨酸各自与天冬氨酸或者谷氨酸相邻时，会引起胰蛋白酶的漏切。此外，还发现组氨酸 赖氨酸 异亮氨酸/ 丙氨酸结构也有此现象。本研究中检测出漏切肽段并没有明显提高序列覆盖率，但显著增加了蛋白质鉴定结果的确定性。在实际样品分析中如将漏切数设置为1有可能产生肽段鉴定的假阴性。

Identification of Tryptic Peptides with Double Missed Cleavage Sites by Mass Spectrometry

To investigate the influence of missed cleavage numbers of trypsin for database searching results, Cytochrome C and abscisic stress ripening-like protein were identified by MALDI-TOF/TOF mass spectrometry. Two and four tryptic peptides with double missed cleavage were found in the digests of cytochrome C and of abscisic stress ripening-like protein, respectively. It was found that two missed cleavage will happe when two lysine residues neighbor to each other or lysines and arginines neighbor with an aspartic acid or glutamic acid residue. It was observed that this phenomenon was related to histine-lysine-isoleunine/alanine sequence. In this study, the certainty of protein identification rather than protein coverage was significantly improved when tryptic peptides with double missed cleavage sites were identified, which indicate that false negative of peptide identification will happen when the number of missed cleavage site was set to 1.